The Position of Circulating Tumor DNA in the Clinical Management of Colorectal Cancer

Simple Summary Currently, methods including endoscopy, radiology, and carcinoembryonic antigen levels allow for the detection of colorectal cancer (CRC) at an early stage and the ability to follow the evolution of the disease during treatment. However, these are not always sensitive and specific enough for timely intervention. This leads, amongst other consequences, to delays in treatment or even to overtreatment. Circulating tumor DNA (ctDNA) has shown promise in filling this gap, allowing treatment to be personalized at each stage of the disease and, thus, tailored to each patient’s needs. This review article focuses on the current clinical use and future direction of ctDNA for CRC management. Abstract Colorectal cancer (CRC) is the third most common cancer type worldwide, with over 1.9 million new cases and 935,000 related deaths in 2020. Within the next decade, the incidence of CRC is estimated to increase by 60% and the mortality by 80%. One of the underlying causes of poor prognosis is late detection, with 60 to 70% of the diagnoses occurring at advanced stages. Circulating cell-free DNA (ccfDNA) is probably the most promising tool for screening, diagnosis, prediction of therapeutic response, and prognosis. More specifically, the analysis of the tumor fraction within the ccfDNA (circulating tumor DNA, ctDNA) has great potential to improve the management of CRC. The present review provides an up-to-date and comprehensive overview of the various aspects related to ctDNA detection in CRC.


Circulating Tumor DNA: Detection
Circulating cell-free DNA (ccfDNA) was first described in human plasma by Mandel and Métais in 1948 [1] and has been found to originate from various cell types, including cancer cells (ctDNA). Increased ccfDNA concentrations have been observed in situations of cell lysis and turnover, including pregnancy, intensive exercise, inflammation, infection, autoimmune diseases, diabetes, or soft-tissue injury [2][3][4][5]. The first connection between ccfDNA and cancer was made in 1977 by Leon et al. through the observation that ccfDNA concentrations were also increased in various types of cancers [6].
The above finding is also reflected in colorectal cancer (CRC). The rate of circulating tumor DNA (ctDNA) detection is highly variable across studies and ranges between 40 and 100% for localized tumors to nearly 100% in metastatic CRC [7,8]. ctDNA comprises a fraction of the total DNA circulating freely in the bloodstream and its proportion within ccfDNA depends on the cancer stage and ranges from 0.01 to 0.0001% to more than 50% [9]. An alternative option consists of non-targeted (agnostic) methods. Indeed, it is possible to fish for alterations that frequently occur in CRC without baseline tumor tissue analysis. A non-targeted approach implies the use of optimized methods able to screen a large number of alterations concomitantly. Recent dPCR techniques (including droplet-based and microfabricated compartment-based platforms) can also be used, allowing high multiplexing through two to six-color detection [19][20][21]). For example, this technique can be used for methylation assays [22]. Optimized NGS methods including Safe-Sequencing System (Safe-SeqS), CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), integrated Digital Error Suppression-enhanced CAPP-seq (iDES-enhanced CAPP-seq), and Base-Position Error Rate (BPER) have a high sensitivity. Nevertheless, they are more expensive and time-consuming than dPCR [23][24][25][26]. Non-targeted approaches have also been developed using Whole Exome Sequencing (WES) [27] and Whole Genome Sequencing (WGS) [28], allowing a complete genotyping and detection of de novo mutations but with a lower sensitivity and higher cost. In non-targeted approaches, the absence of detection could mean that the alterations were either absent in the blood or in the tumor itself [3].
Overall, detection methods must be selected according to the sampling conditions and the purpose of the studies [29]. For clinical applications, the most favorable test is timeand cost-effective with acceptable sensitivity and specificity.

Delay of Sampling Delay between Surgery and ctDNA Sampling
The best timing for ctDNA sampling is controversial, and only a few publications have been dedicated to this topic. In non-metastatic CRC, a drop in ccfDNA concentration immediately after surgical treatment has been reported [30]. The ccfDNA rises beginning at 24 h after surgery and can be used to discriminate patients with recurrence after 48 h [30]. However, early blood collection (before week 4) may theoretically reduce the sensitivity of ctDNA detection because of ccfDNA release as a consequence of the surgical trauma [31]. In the study by Scholer et al., blood samples were collected on day 8, day 30, and every month. Interestingly, out of 26 operated patients, 2 were ctDNA+ eight days after surgery, and 2 others became ctDNA+ one month after surgery [32]. Overall, collecting blood early after surgery might be more relevant for immediate clinical application but must be weighed against a higher rate of false negatives. Figure 1 illustrates some applications of ctDNA monitoring before and after treatment in various settings. This section will describe the significance of ctDNA as a screening, diagnostic, prognostic, predictive, minimal residual disease, and recurrence marker.

ctDNA for Early Cancer Detection: Screening
Because survival is highly affected by the stage at diagnosis, early detection of CRC is critical [33]. Most screening programs for CRC are currently based on a non-invasive stoolbased test, either the guaiac-based fecal occult blood test (gFOBT) or the immunological fecal occult blood test (iFOBT), also referred to as the fecal immunochemical test (FIT). These tests are not specific and present a low sensitivity for the detection of CRC. When positive, CRC must be confirmed by a complete colonoscopy, which is invasive, expensive, and often requires sedation [33][34][35][36]. ing blood early after surgery might be more relevant for immediate clinical application but must be weighed against a higher rate of false negatives. Figure 1 illustrates some applications of ctDNA monitoring before and after treatment in various settings. This section will describe the significance of ctDNA as a screening, diagnostic, prognostic, predictive, minimal residual disease, and recurrence marker. Figure 1. Clinical relevance of ctDNA in colorectal cancer. This figure depicts the critical applications in the clinical setting using both the current clinical gold standards and ctDNA. These include tumor genotyping in cancer diagnosis, assessing treatment response, tracking minimal residual disease and relapse, and monitoring clonal evolution. (1) Screening is routinely performed to detect CRC at an early stage using a fecal occult blood test (FOBT), but replacement by ctDNA has yet to be encouraged. (2) Diagnosis is performed before therapy to confirm the tumor's presence. When using ctDNA, genotyping could determine the tumor profile and identify patients with a high tumor burden. Determination of tumor burden has the potential to help guide neo-adjuvant or Figure 1. Clinical relevance of ctDNA in colorectal cancer. This figure depicts the critical applications in the clinical setting using both the current clinical gold standards and ctDNA. These include tumor genotyping in cancer diagnosis, assessing treatment response, tracking minimal residual disease and relapse, and monitoring clonal evolution. (1) Screening is routinely performed to detect CRC at an early stage using a fecal occult blood test (FOBT), but replacement by ctDNA has yet to be encouraged. (2) Diagnosis is performed before therapy to confirm the tumor's presence. When using ctDNA, genotyping could determine the tumor profile and identify patients with a high tumor burden. Determination of tumor burden has the potential to help guide neo-adjuvant or adjuvant therapy and monitor response. (3/6) Detection of residual disease and recurrence is performed using radiologic imaging and carcinoembryonic antigen (CEA) detection. However, the former suffers from a delay in detection and the latter suffers from a lack of sensitivity. Assessment of ctDNA after therapy facilitates the detection of both emerging resistance mutations and minimal residual disease (MRD) before progression, with the potential for the non-invasive prediction of recurrence. (4/5) Guiding treatment and monitoring treatment response occurs based on the presence or absence of tumor lesions. On the other hand, ctDNA can guide genotype-directed therapy and allows for the monitoring of the response to treatment based on tumor burden. When acquired resistance to targeted therapies occurs, ctDNA can detect specific mechanisms or resistance, considering the different clones present within the primary tumor and all metastatic sites, and can guide treatment adjustments. In contrast, imaging and the CEA marker can detect the emergence of resistance without knowing the mechanisms of resistance. Created with BioRender.com.

ctDNA: Clinical Applications
To circumvent these limitations, ctDNA has been explored as a potential screening tool for CRC. Table 2 shows a comparison of the different CRC screening methods. Taking advantage of the fact that aberrant DNA methylation is generally one of the first steps in CRC carcinogenesis, several methylation signatures have been explored [37] using one [38] or multiple gene methylation profiles [39,40]. Amongst various methods, the best sensitivity (75-81%) and specificity (96-99%) so far have been provided by the Epi ProColon ® 2.0 test (Epigenomics AG, Berlin, Germany) which is based on the detection of hypermethylation on the SEPT9 promoter [38]. To date, this test is the only blood-based qualitative screening test accepted by the FDA for CRC. In a population of 7941 asymptomatic individuals (PRESEPT), the SEPT9 detected CRC with sensitivities of 35.0%, 63.0%, 46.0%, and 77.4%, in stages I to IV, respectively. In this population, the sensitivity for advanced adenomas was only 11.2% [41] which remains insufficient to replace standard colonoscopy screening. However, one of the main advantages of the SEPT9 test is the increased patient compliance compared to the colonoscopy [42,43]. The Epi ProColon ® 2.0 test aims explicitly at detecting CRC, but early detection tests are being developed that target a variety of cancers, including CRC. GRAIL Inc. recently published a novel test (Galleri ® test) for the early detection of more than 50 types of cancer simultaneously, including CRC. This test analyses specific methylation patterns in ccfDNA that have been associated with many cancer entities. The tissue of origin can be predicted with 96% specificity and 93% accuracy. Moreover, the sensitivity in all cancer types was 18% (stage I), 43% (stage II), 81% (stage III), and 93% in stage IV. This corresponds to the sensitivity in detecting CRC, approximately 28% (stage I), 70% (stage II), 78% (stage III), and 97% (stage IV). These results indicate an important potential benefit to using methylated ccfDNA for cancer detection [44]. In general, the concentration of ctDNA in patients' blood at early stages is low or nonexistent, making its detection challenging for screening purposes. The American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) has concluded that there is still little evidence for the clinical validity of ctDNA detection for cancer screening [46]. However, ctDNA detection could be combined with traditional screening methods to improve the diagnosis of CRC at an early stage [47][48][49].
In the non-metastatic setting, the prognostic value of ctDNA detection at baseline (i.e., before surgery) is unclear. In the study of Reinert et al., almost all patients (stage I-III) presented with ctDNA+ detection before surgery, which was not associated with the risk of recurrence [8]. In the ALGECOLS (Presence of Circulating Tumour DNA in Colorectal Cancer) study (NCT01198743), 27.5% of the patients were ctDNA+ before surgery. These ctDNA+ patients showed a higher rate of recurrence (32.7% versus 11.6% in ctDNA− patients, p = 0.001). In addition, the time to recurrence (TTR) was significantly shorter in  [56]. Those discrepancies suggest that survival is probably associated with ctDNA concentration rather than a simple 'yes or no' detection. In patients with metastatic CRC, the tumor load before treatment is an important prognostic factor [58][59][60]. The ctDNA concentration before chemotherapy administration can be considered a continuous variable, with the highest concentrations being associated with the shortest survival [61].

ctDNA as a Prognostic Biomarker in CRC Stage I-III: Detection of Minimal Residual Disease
After curative-intent surgery, adjuvant chemotherapy (ACT) is routinely delivered to patients with high-risk stage II or stage III CRC. However, >50% of stage III and >80% of stage II patients are exposed to unnecessary chemotherapy. In fact, the 5-year DFS rate of stage II and low-risk stage III patients who underwent surgery alone has been reported as 78-91% and 78%, respectively [62]. Since the treatment is associated with lifetime side effects (e.g., chemotherapy-induced neuropathy), one aims to reduce the incidence of ACT when proven unnecessary [63].

Detection of MRD/Recurrence after Surgery
In a pioneering study of 230 patients with stage II colon cancer, Tie et al. investigated the ability to identify patients at high risk of recurrence by detecting post-operative ctDNA [64]. The delay for post-operative plasma withdrawal was 4 to 10 weeks. The rate of ctDNA-positive detection was 8.7% for the whole cohort. In patients not treated with ACT (n = 178), ctDNA was detected post-operatively in 7.9% and was associated with a 79% recurrence at a median follow-up time of 27 months. The recurrence occurred in only 9.8% of patients with negative ctDNA (HR 18; 95% CI, 7.9 to 40; p < 0.001). In this study, 52 patients with histological high-risk stage II CRC were treated with ACT. Among them, six were positive for ctDNA detection after surgery, and three recurred despite the adjuvant treatment [64].
Using a tumor-informed Safe-SeqS platform, Tie et al. further analyzed the ctDNA status in a cohort of patients with stage III CRC [50]. The ctDNA was detectable in 21% of the cohort 4 to 10 weeks after surgery. A recurrence was observed in 42% of the patients with post-operative ctDNA+.
In a longitudinal cohort study, ctDNA was used to monitor tumor burden in 21 CRC patients (stages I-III) who underwent ctDNA analysis three months after complete surgery. In all six patients with detectable ctDNA, the recurrence occurred within three years. In patients without detectable ctDNA, the recurrence rate was 27% (4/15) {HR, 37.7; 95% CI, 4.2-335.5; p < 0.001} [32].
Mixing a population of patients with stage II and III, Li et al. found 27.8% disease progression for ctDNA-positive patients after surgery (ctDNA sampling within one week after surgery and follow-up of 6 months) compared to 4.4% for those who were ctDNAnegative (Fisher test, OR 7.9, p = 0.0169, 95% CI) [65].
Similarly, Benhaim et al. evaluated the pertinence of longitudinal detection and quantification of ctDNA prospectively as a prognostic marker of recurrence in the ALGECOLS (Presence of Circulating Tumor DNA in Colorectal Cancer) study (NCT01198743). The ctDNA analysis was performed before and after surgery in 184 patients (stage II-III) during 3-4 years of follow-up using ddPCR. After surgery, 18/171 (10.5%) patients were ctDNA+. Positive ctDNA levels after surgery were associated with a 44.4% recurrence rate versus 13.7% in ctDNA− patients (p = 0.003) [51].
Chen et al. also found that post-operative serial ctDNA detection predicted a high risk for recurrence. Low recurrence risk was observed in ctDNA− patients, with a 2-year RFS rate of 89 The abovementioned studies that have shown the relevance of ctDNA as a marker for the detection of MRD are listed below in Table 4.

ctDNA Clearance after Treatment
At each stage of treatment, variations in ctDNA concentration and ctDNA clearance might reflect treatment efficacy. However, the rate of ctDNA clearance has not been evaluated because of the lack of extensive studies with longitudinal sampling. The main results are summarized in Table 5.  In stages II-III CRC, the ctDNA status changed from positive to negative in 75-92% of the patients after surgery [51,56].
Rate of ctDNA clearance after adjuvant chemotherapy: In stages I-III CRC, the rate of ctDNA clearance observed under chemotherapy was between 50% and 68%. This rate is approximately the same for stage II and stage III CRC.

•
In stages I-III, Reinert et al. observed 30% of patients who cleared ctDNA after ACT and stayed disease free throughout the study [8].

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In stage II, Tie et al. observed that post-operative ctDNA+ remained negative after ACT in three out of six patients [64].

•
In stage III, the ctDNA status changed from positive to negative in 50-68% of the patients after completion of chemotherapy treatment [50,51,67].
ctDNA clearance is associated with a superior RFS in most series: In stages II and III, superior RFS was observed when ctDNA became undetectable after chemotherapy (HR 5.11; p = 0.02) [64]. The absence of ctDNA clearance after chemotherapy is associated with a rate of 30% RFI at three years (HR, 6.8; 95% CI, 11.0-157.0; p < 0.001) [50].

Value of ctDNA in the Prediction of Relapse before Conventional Imaging Techniques
Predicting relapse before radiologic recurrence is necessary. ctDNA detection can anticipate radiological recurrence with a lead time of 3 to 12 months, as described in recent publications (see Table 6). These results are clearly subject to bias, as the usual interval between plasma sampling is three months, whereas, for imaging assessments, it is six months. Although this anticipation is crucial, we should be aware that false-positive results exist and overall survival has not been shown to increase with the earlier treatment of relapses. The French trial CIRCULATE-MRD has recently been funded and will soon open for inclusion to address this question. In addition, the sensitivity and specificity of ctDNA detection during follow-up should be further studied to determine whether longitudinal sampling could be a way to avoid or delay (reduce) imaging follow-up. The ctDNA concentration varies throughout the treatment duration; it reflects a response to treatment and allows for the selection of non-responding patients. Table 7 summarizes the main studies that have addressed this knowledge area.  Early changes in ctDNA concentration during the treatment course predict subsequent radiologic responses, suggesting that ctDNA is a marker of therapeutic efficacy [61,74,75]. Moreover, in some studies, changes in ctDNA also affected PFS and OS, where patients with relatively low ctDNA concentrations showed longer PFS and OS compared to patients with higher ctDNA concentrations [61].
Overall, the longitudinal surveillance of ctDNA allows for the early detection of relapse and response to intervention [32,76]. The detection of ctDNA could participate in the individual management of patients based on their tumor's genetic profile, as the behavior of cancer in response to therapy can be predicted by determining ctDNA concentrations [29].

ctDNA Predicts Response to Targeted Therapy
A prime example concerns the eligibility for anti-EGFR treatment, where the RAS mutation status of tumor tissue must be determined prior to formulating a treatment plan [17,18]. Since the concordance level in KRAS mutational status between tumor tissue and ctDNA is high (∼92%) [77], the detection of KRAS mutations in ctDNA has been proposed as a rapid and minimally-invasive alternative method to tissue biopsy for predicting the response to anti-EGFR treatment [78]. Interestingly, KRAS mutations have also been detected in ctDNA, although the primary tumor was considered wild-type. These circulating mutations may reflect the existence of minor cell subclones in the primary tumor or its related metastases [5]. Knebel et al. described the monitoring of a patient with KRAS wild-type mCRC treated with chemotherapy combined with anti-EGFR therapy [79]. Surprisingly, KRAS mutations in ctDNA were detected after the first exposure to anti-EGFR therapy but before clinical progression. Subsequently, the evolution of the disease went along with increasing concentrations of KRAS-mutated ctDNA. These results support the importance of the longitudinal monitoring of KRAS mutations in ctDNA before and during anti-EGFR therapy for the early detection of increasing cell clones that could be associated with drug resistance [79].
The emergence of RAS mutations in initially RAS wild-type tumors is a well-known mechanism of acquired resistance to anti-EGFR therapy. Nevertheless, whether these mutations are acquired de novo or whether initially undetectable mutant subclones proliferate through clonal selection and evolution remains unclear [80,81]. A subsequent treatment involving the withdrawal of EGFR blockade may be followed by an increase in the proportion of wild-type (sensitive) clones and a decrease in resistant (RAS mutant) clones, even to undetectable levels [81]. This work laid the foundation for the activity of anti-EGFR rechallenge. The CRICKET (Cetuximab Rechallenge in Irinotecan-Pre-treated mCRC, KRAS, NRAS, and BRAF wild-type Treated in 1st line With Anti-EGFR Therapy) trial (NCT02296203) demonstrated that a rechallenging strategy (in a third-line setting) with cetuximab and irinotecan can be effective, whereby evaluating the RAS mutation status on ctDNA might help select candidate patients and guide therapeutic decisions [71]. Patients with RAS wild-type ctDNA had a significantly longer PFS than those with RAS mutated ctDNA (median PFS: 4.0 vs. 1.9 months; hazard ratio: 0.44; 95% CI, 0.18-0.95; p = 0.03) [71].
Several preclinical studies have suggested that ERBB2 (HER2) copy number gain is a negative predictor of response to anti-EGFR therapy [82]. Investigators of the HERACLES A study, a phase II trial of trastuzumab and lapatinib in chemotherapy and EGFR antibodyrefractory HER2-positive mCRC patients, reported that ctDNA precisely predicted the response to anti-HER2 therapy in HER2-positive CRC [72]. In total, 47 of 48 samples from 29 patients had detectable ctDNA, and 46 out of 47 samples were HER2-positive {2.55-122 copies; 97.9% sensitivity (95% CI, 87.2-99.8%)}. These results support the use of adequately validated ctDNA testing as an alternative to tissue biopsy to identify individuals who may benefit from anti-HER2 therapy [72].
Herbst et al. suggested that detecting HPP1 methylation in ctDNA could be used as an early marker to identify patients likely to benefit from a combination of chemotherapy and bevacizumab [73]. Before starting treatment, 337 of 467 patients had detectable methylated HPP1 ctDNA. Two to three weeks after starting treatment, methylated HPP1 ctDNA levels decreased to undetectable levels in 167 of 337 patients. These patients showed improved OS compared to patients with continued detection of methylated HPP1 ctDNA. In addition, methylated HPP1 ctDNA is predictive for combination therapy as early as 3 weeks after the start of treatment, whereas radiological imaging cannot do so until 12 or 24 weeks [73]. Table 8 lists several ongoing trials using ctDNA to guide treatment. In the nonmetastatic setting, ways to better select adjuvant treatment are actively sought. For example, the ongoing ctDNA-guided single-arm phase II CHRONOS (Rechallenge With Panitumumab Driven by RAS Clonal-Mediated Dynamic of Resistance) trial (NCT03227926) aims to determine which patients are eligible for anti-EGFR rechallenge. This study uses the ctDNA analysis of RAS, BRAF, and EGFR mutations to drive anti-EGFR rechallenge therapy in mCRC [83]. Based on the same model, second-line rechallenge with cetuximab is under evaluation in the CRICKET trial [71].

ctDNA as a Tool for Guiding Treatment
In the metastatic setting, several studies have assessed the accuracy of ctDNA-based genotyping in selecting patients for mutation-directed therapy [84][85][86]. Currently, the ongoing prospective, multicentric interventional study (Following Therapy Response Through Liquid Biopsy in Metastatic Colorectal Cancer Patients, FOLICOLOR) in Belgium is evaluating the utility of ctDNA genotyping to monitor clinical response and guide therapeutic decision-making. In patients with unresectable metastatic disease, progressive disease is identified by NPY methylation in ctDNA. Two primary endpoints are (1) investigating whether ctDNA can detect progressive disease earlier than conventional monitoring based on CT imaging and (2) whether adapting treatment based on ctDNA could improve progression-free survival and overall survival.   [52] Control arm: surveillance according to current Danish Guidelines with CT-scans at 12-and 36-months post-operative and colonoscopy every 5 years until age 75 Experimental arm: ctDNA-guided surveillance every 4 months postoperatively.
(2) ctDNA-negative: high-intensive radiological surveillance with FDG-PET/CT-scan every 3 months until recurrence detection or 21 months have passed Fraction of patients with relapse receiving curative-intended resection or local treatment

Rectal Cancer: The Current State of Management
Approximately one-third of all newly diagnosed CRC is composed of rectal cancer. Currently, the standard treatment for advanced rectal cancer consists of neoadjuvant radiotherapy, with or without sensitizing chemotherapy, followed by surgery with total mesorectal excision (TME) [94,95]. Locally advanced rectal cancer (LARC) is exceptionally challenging to manage, given the structural constrictions of the pelvis. Due to the anatomical challenges encountered during TME, there is an increased risk of operative morbidity and mortality and sexual, urinary, and bowel dysfunction [96].
For smaller tumor lesions and under specific conditions (mostly <4 cm, ≤T3), the neoadjuvant treatment allows avoiding TME for 20-30% of LARC patients who achieve a clinical complete response (cCR) [97,98]. However, the risk of local recurrence and distant metastases remains present within this patient population [99]. Despite improvements in pre-operative care and surgical techniques, the quality of life and survival rates remain subpar among rectal cancer patients. Selecting patients who may most benefit from conservative treatment is crucial.
Other studies are currently investigating the significance of ctDNA in directing nonoperative management approaches for LARC patients, as shown in Table 10.

Conclusions and Perspectives
This review provides an up-to-date and comprehensive summary of the various areas of ctDNA research in CRC management. As this review points out, numerous studies have shown the association of ctDNA with tumor burden and its usefulness in detecting and monitoring tumor dynamics, drug response, and resistance to therapy with increasing sensitivities and specificities.
The experimental detection methods are numerous and of great importance in the interpretation of study results. Further improvement in the standardization of methods leading to the preanalytical variability of liquid biopsies is imperative to obtain optimal sensitivity and specificity for the reliable use of ctDNA in daily practice.
At this point, ctDNA detection has yet to be accepted as a worthwhile CRC screening tool. The low concentration available in the early stages imposes the use of highly-sensitive tests, which are currently cost-prohibitive for routine use. The cost of a ctDNA detection assay ranges from EUR 168 to EUR 1423 per sample in a maximum-testing condition, as is expected in future standard practice [111]. Despite these current high costs, active research to improve the methodology and reduce costs is underway. These efforts are driven by the marked advantages of ctDNA, including its accuracy, ease of collection, and minimal invasiveness. It is precisely for this reason that large-scale clinical trials are underway to explore how to optimize ctDNA detection alone or in combination with conventional screening methods.
After surgery, ctDNA can clearly identify patients at low and high risk of relapse, which has direct implications for adjuvant therapy decisions. In the French multicenter adjuvant trial, CIRCULATE-PRODIGE 70, the administration of adjuvant therapy in patients with stage II CRC is based on post-operative ctDNA detection [87]. The ongoing Tracking Mutation in Cell Free Tumor DNA to Predict Relapse in Early Colorectal Cancer (TRACC) study aims to compare ctDNA versus standard of care in predicting relapse in patients with stage II and III CRC undergoing ACT after surgery [90]. In this setting, highly-sensitive tests are critical to avoid both over-and undertreatment.
In patients with metastatic CRC, serial ctDNA testing provides early indications of the clinical efficacy of therapy. In this setting, the variation in ctDNA concentration is related to the response to systemic treatments. Future clinical trials incorporating ctDNA concentration into the study design may allow for the real-time measurement of therapeutic efficacy. Serial testing is also used to validate ctDNA as a detection method of recurrence. One example is the IMPROVE-IT2 (Implementing Noninvasive Circulating Tumor DNA analysis to Optimize the Operative and Post-operative Treatment for Patients with Colorectal Cancer-Intervention Trial 2) trial. This randomized controlled trial investigates the benefit of ctDNA-guided post-operative surveillance compared to the current standard-of-care CT imaging-based surveillance. The main objective is to investigate whether ctDNA-guided surveillance increases the proportion of patients with recurrence receiving curative-intended resection or local metastasis-directed treatment [91].
As data from the many ongoing clinical trials of ctDNA in CRC emerge, better guidelines will arise on incorporating ctDNA into clinical decision-making. Nevertheless, given the heterogeneous nature of colorectal tumors, a single biomarker might be insufficient for managing CRC. Biomarkers could be combined in composite panels such as protein biomarkers, circulating tumor cells, micro RNAs, and ctDNA.
Altogether, evidence strongly indicates that ctDNA should be considered a key tool in the implementation of a personalized medicine approach; it is only a matter of time before ctDNA becomes a crucial part of clinical medicine.

Conflicts of Interest:
The authors declare no conflict of interest.